WO2004045561A2 - Use of ferritin for immunomodulation - Google Patents

Abstract

The invention relates to the discovery that ferritin is involved in the introduction of B cell proliferation, maturation, and differentiation. The invention includes compounds that modulate ferritin expression or activity. The invention also encompasses the use of ferritin to induce B cell proliferation, maturation, and differentiation.

Description

Use of Ferritin for Immunomodulation

TECHNICAL FIELD

This invention relates to immunomodulation, in particular, immunomodulation of B lymphocytes.

BACKGROUND

A syndrome of B lymphocyte dysfunction has been described in Human Immunodeficiency Virus (HIV) infection. The syndrome includes the hyperactivation of B cells leading to their proliferation (Schnittman et al., 1986, Science 233:1084- 1086), hypergammaglobulinaemia, the unregulated, over-production of immunoglobulin (Ig) of IgA, IgG and IgM isotypes (Katz et al., 1986, Clin. Immunol. Immunopathol. 39:359-367; Reimer et al., 1988, Monogr. Allergy 23:83-96; Uko et al., 1994, Afr. J. Med. Med. Sci. 23:385-389), and the loss of an immune response to recall antigens. For example, immunological reactivity acquired by prior vaccination to tetanus toxoid is greatly diminished, yet the response to polyclonal or mitogenic antigen is retained (Katz et al., 1986, Clin. Immunol. Immunopathol. 39:359-367). Several co-factors have been proposed as mediators of B cell dysfunction in HIN infection including the viral envelope glycoprotein gpl20, tumor necrosis factor α (TΝPα; Patke and Shearer, 2000, J. Allergy Clin. Immunol. 105:975-982), HIN- dependent upregulation of interleukin-6 (IL-6; Rautonen et al., 1991, AIDS 5:1319- 1325), interleukin-10 (IL-10; Mϋller et al., 1998, Blood 92:3721-3729), or interleukin-15 production (IL-15; Kacani et al., 1997, Clin. Exp. Immunol. 108:14- 18). However, evidence that these proposed mediators account for all three facets of HIV-dependent B lymphocyte dysfunction in vitro or in vivo is incomplete. At best, hyperactivation and the induction of immunoglobulin synthesis has been ascribed directly or indirectly to these proposed mediators, but correlation to patient data is poor and none of these factors have been reported to abrogate B cell responsiveness to recall antigen.

B cell maturation takes place initially in the bone marrow where mature, but antigen naive cells are produced. During this antigen-independent phase, B lymphocytes undergo immunoglobulin gene rearrangement to express surface-linked IgM and IgD before emergence into the peripheral system. Only approximately 10% of precursor B lymphocytes complete this process. These lymphocytes have a limited life unless they encounter soluble antigen; either T cell-independent or T cell- dependent with activated T helper cells. B cells then enter an antigen-dependent maturation where the cell receives two successive signals to activate and proliferate, a competence signal and progression signal. Depending upon the exact nature of the subsequent signals from accessory cells, B lymphocytes undergo affinity maturation and class-switching of immunoglobulin genes and differentiation into antibody secreting plasma cells. Concomitantly, a population will adopt a resting, memory cell phenotype and persist within the body providing the basis for future response to the antigen.

Ferritin is a ubiquitous iron storage protein found in all tissues of the body, as well as serum and is principally associated with iron metabolism, storage, and detoxification. Multiple forms of ferritin containing combinations of two protein species exist, referred to as the basic or light chain and acidic or heavy chain in large 24 subunit homo or hetero-polymers which can contain up to 4,500 molecules of inorganic iron (Harrison and Arosio, 1996, Biochim. Biophys. Acta 31:161-203). Elevated serum ferritin levels can be a marker of patient ill health. In HIN and AIDS, increased serum ferritin levels are associated with a poor prognosis (Ellaurie and Rubinstein, 1994, Acta Paediatr. 83:1035-1037; Lane et al., 1983, Ν. Engl. J. Med. 309:453-458; Savarino et al., 1999, Cell Biochem. Funct. 17:279-287).

SUMMARY

The present invention is based, in part, on the discovery of previously unknown immunological properties of ferritin and the identification of ferritin as an important target for therapeutic intervention during HIV infection. It has been discovered that ferritin is involved in HIV-1 replication and facilitation of the B lymphocyte dysfunction that is observed during HIV-1 infection. Also, it has been found that the release of ferritin from HIN-1 infected primary macrophages is dependent on the accessory protein Νef. Analysis of the Νef-dependent release of ferritin revealed previously unknown B cell immunomodulatory functions of ferritin. Accordingly, the invention relates to the previously unknown ability of ferritin to stimulate B lymphocytes, addition, ferritin represents a therapeutic target for treating HIV infection and HIV-associated lymphoma. The immunomodulatory properties of ferritin also have applications for its use as an adjuvant to vaccine preparations designed to enhance an antibody response. Accordingly, the invention relates to a method of increasing at least one of B lymphocyte proliferation, differentiation, or maturation. The method includes administering ferritin (e.g., an L subunit of ferritin, either alone or in combination with an H subunit) or a compound that increases ferritin expression or activity to a B lymphocyte or a B lymphocyte precursor cell in an amount sufficient to increase B lymphocyte proliferation, differentiation, or maturation. In some embodiments, the compound is Nef or an active fragment thereof. The compound may be administered to a cell or a plurality of cells in vitro. The cell can be a primary cell, a secondary cell, or an immortalized cell. The ferritin or compound can be naturally occurring or recombinant (e.g., a recombinant L subunit). In some embodiments, the production of at least one of IgG, IgA, or IgM is increased. In another aspect, the compound is administered to a subject (e.g., a mouse or a human). The subject may have a disorder associated with B cells, e.g., Burkitt's lymphoma or Hodgkin's lymphoma. Alternatively, the compound may be administered to a cell in vitro. In some cases, the cell is genetically engineered, e.g., to produce a recombinant antibody. Included is a method for identifying a compound that inhibits the activity of ferritin on a lymphocyte (e.g., a peripheral blood monocyte or a B lymphocyte). The method includes the steps of obtaining a cell sample containing a lymphocyte (e.g., a peripheral blood monocyte or a B lymphocyte), incubating the cell sample in the presence of ferritin and a test compound, and determining whether the test compound modulates a ferritin-associated effect on the lymphocyte in the presence of ferritin compared to a cell sample incubated in the absence of the test compound and in the presence of ferritin. In some embodiments the compound decreases the ferritin- associated effect on the lymphocyte. In an alternative embodiment, the compound increases the ferritin-associated effect on the lymphocyte. The compound may decrease the ferritin-associated effect on the lymphocyte and decrease the expression or activity of NF-κB, BCL-2, Casper/c-FLEP, or cyclin Dl; or the compound increases the ferritin-associated effect on the lymphocyte and increases the expression or activity of NF-κB, BCL-2, Casper/c-FLIP, or cyclin Dl. In some cases, the compound increases the ferritin-associated effect on the lymphocyte and decreases expression or activity of a cell cycle progression protein, e.g., p27^KIP1 or p53. In some embodiments, the compound does not affect MEK1 expression or activity. In some aspects, the ferritin-associated effect on the lymphocyte is at least one of the following: cellular proliferation, differentiation into a plasma cell, increased secretion of non-specific immunoglobulin, increased secretion of IgG, increased secretion of IgM, secretion of IgA, increased secretion of non-specific immunoglobulin, increased expression of IgG, increased expression of IgM, and increased secretion of IgA. In some cases, the invention relates to a method of decreasing B lymphocyte proliferation, differentiation, or maturation. The method includes contacting a B lymphocyte or precursor of a B lymphocyte with compound that inhibits ferritin expression or activity in an amount sufficient to decrease B lymphocyte proliferation, differentiation, or maturation. In some embodiments the compound inhibits expression or activity of at least one of Nef or NF-κB. In some cases, the compound does not not affect MEK1 expression or activity. The compound can be administered to a cell that is in vitro or a cell that is in a mammal such as a mouse or human. The cell can be a B lymphocyte or precursor of a B lymphocyte, and the cell can be from a subject who has Burkitt's lymphoma or Hodgkin's lymphoma. Also provided is a method of inhibiting immunoglobulin expression in a B cell. The method includes providing a B cell and contacting the cell with an inhibitor of ferritin expression or activity. The cell can be from a mammal, e.g., a mouse or a human, and can be in vitro or in a mammal such as a mouse or human.

A B lymphocyte-related disorder is a disorder in which there is either undesirable proliferation of B lymphocytes (e.g., B cell-associated lymphomas) or a disorder in which it is desirable that there be increased B lymphocyte proliferation (e.g., in certain disorders associated with immunosuppression). This includes disorders in which treatment is related to the suppression of proliferation or the induction of proliferation of a specific clonal line of B cells. Disorders include HIN infection in which, e.g., macrophage release of ferritin induces abnormal B lymphocyte proliferation and secretion of immunoglobulins. Also, other viral, parasitic (e.g., Chagas disease), and bacterial infections can be associated with undesirable B cell activation.

Also included are compositions that include the cells or compounds disclosed herein. A "subject" is a mammal, e.g., a human, or can be an experimental or animal or disease model. The subject can also be a non-human animal, e.g., a non-human primate, mouse, rat, horse, cow, goat, or other domestic animal.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the detailed description, drawings and from the claims.

DESCRIPTION OF DRAWINGS

Fig. 1 A is a bar graph showing the results of experiments measuring ³H- thymidine incorporation into purified B cells or T cells incubated in the presence of cell culture supernatant of macrophages expressing Nef (Adeno-Nef), GFP (Adeno- GFP), or mock infected.

Fig. IB is a bar graph showing the results of experiments measuring H- thymidine incorporation into B cell controls (for the experimental results depicted in Fig. 1 A) incubated in medium or medium supplemented with anti-IgD (25 μg/ml) +rCD40L (50 ng/ml, Alexis Corporation, San Diego, CA) +IL-4 (10 ng/ml).

Fig. 2A is a diagram of ferritin light chain subunit gene and protein sequence (SEQ ID NO: 1).

Fig. 2B is a photograph of a non-denaturing SDS-PAGE of highly concentrated macrophage supematants stained with Commassie Blue and the microsequencing results for band 1 (SEQ ID NOS:2-8) and band 2 (SEQ ID NOS:9- 13).

Fig. 2C is a bar graph depicting the results of experiments measuring the amount of ferritin released from adenovirus transduced macrophages expressing Nef, GFP, or mock infected.

Fig. 2D is an image of a Western blot of concentrated macrophage supernatant stained with anti-ferritin antibody. The supematants were prepared from medium in which macrophages infected with Adeno-Nef, Adeno-GFP, mock infected were cultured as well as supernatant prepared from medium in which untreated macrophages were cultured and medium from macrophages grown in serum-free medium, and purified liver ferritin.

Fig. 3 A is a graph showing the growth of macrophages infected with HIV-1, HIN-1 containing a defective nef gene (Delta nef or Δnef); or mock infected. ADA is the strain of HIV-1 used. RT is reverse transcriptase activity (an assay for the growth of HIV; results are expressed as counts per minute (cpm) per 10 μl).

Fig. 3B is a graph showing the results of experiments in which the release of ferritin from macrophages infected with HIV-1, HIV-1 having a defective nef gene (Δnef), or mock infected.

Fig. 3C is a graph showing the results of experiments in which the release of macrophage-colony stimulating factor (M-CSF) from macrophages infected with HIN-1, HIV-1 having a defective nef gene (Δnef), or mock infected.

Fig. 4A is a bar graph depicting the results of experiments measuring IgM release from B cells incubated in supematants from macrophages infected with HIN-1, HIV-1 having a defective nef gene (Δnef), or mock infected. Fig. 4B is a bar graph depicting the results of experiments measuring IgG release from B cells incubated in supematants from macrophages infected with HIV-1, HIN-1 having a defective nef gene (Δnef), or mock infected.

Fig. 4C is a bar graph depicting the results of experiments measuring IgA release from B cells incubated in supematants from macrophages infected with HIN-1, HIV-1 having a defective nef gene (Δnef), or mock infected. Fig. 5 A is a bar graph depicting the results of experiments measuring B cell proliferation after incubation in medium prepared from macrophages infected with adenovirus containing a nef gene, adenovirus containing a GFP gene, mock infected, or ferritin depleted media. The inset table indicates the amount of ferritin present in the supematants after depletion using a non-specific isotype Ig or anti-ferritin Ig.

Fig. 5B is a bar graph depicting the results of experiments measuring immunoglobulin release from B cells after incubation in medium prepared from macrophages infected with adenovirus containing a nef gene, adenovirus containing a GFP gene, mock infected, or ferritin depleted media. The inset table indicates the amount of ferritin present in the supematants after depletion using a non-specific isotype Ig or anti-ferritin Ig.

Fig. 6 A is a graph illustrating the results of experiments detecting the incorporation of ³H-thymidine into peripheral blood lymphocytes incubated in purified ferritin for four days. Fig. 6B is a graph illustrating the results of experiments detecting the incorporation of ³H-thymidine into peripheral blood lymphocytes incubated in recombinant ferritin light chain for four days.

Fig. 6C is a graph illustrating the results of experiments detecting the incorporation of ³H-thymidine into lymphocytes incubated in purified ferritin for four days.

Fig. 6D is a graph illustrating the results of experiments detecting the incorporation of ³H-thymidine into B lymphocytes incubated in recombinant ferritin light chain for four days.

Fig. 6E is a bar graph depicting the results of experiments detecting the incorporation of ³H-thymidine in activated and resting B lymphocytes. rCD40L is recombinant CD40 ligand, which activates B cells and macrophages (Alexis Corporation, San Diego, CA).

Fig. 7A is a graph illustrating the results of experiments detecting the release of IgA from B lymphocytes incubated in ferritin. X axis is the concentration of ferritin in ng/ml.

Fig. 7B is a graph illustrating the results of experiments detecting the release of IgG from B lymphocytes incubated in ferritin Fig. 7C is a graph illustrating the results of experiments detecting the release of IgM from B lymphocytes incubated in ferritin.

Fig. 7D is a bar graph depicting the results of experiments detecting immunoglobulin secretion from B lymphocytes incubated in the presence of anti- human IgD+IL-4+rCD40L.

Fig. 8 A is a bar graph showing the results of experiments detecting plasma ferritin levels in H1N-1 infected individuals.

Fig. 8B is a bar graph showing the results of experiments detecting IgA levels in HIN-1 infected individuals. Fig. 9A is a plot of a flow cytometry analysis of CD38^hl B cells produced by incubation with ferritin (200 ng/ml). Cells were stained with CD138-PE and anti- human IgD-FITC.

Fig. 9B is a plot of a flow cytometry analysis of CD38^h' B cells produced by incubation without additions. Cells were stained with CD138-PE and anti-human IgD-FITC.

Fig. 9C is a plot of a flow cytometry analysis of CD38^hl B cells produced by incubation with pokeweed mitogen. Cells were stained with CD138-PE and anti- human IgD-FITC.

Fig. 10A is a plot of a flow cytometry analysis of CD38^hl B cells produced by incubation with supernatant prepared from medium in which macrophages expressing nef (Ad-Nef) were cultured for four days, washed and cultured for an additional seven days. Cells were stained with CD138-PE and anti-human IgD-FITC.

Fig. 10B is a plot of a flow cytometry analysis of CD38^hl B cells produced by incubation with supematants prepared from medium in which macrophages expressing GFP (Ad-GFP) were cultured for four days, washed, and cultured for an additional seven days. Cells were stained with CD138-PE and anti -human IgD-FITC.

Fig. 10C is a plot of a flow cytometry analysis of CD38^hl B cells produced by incubation with supematants prepared from medium in which macrophages that were mock infected were cultured for four days, washed, and cultured for an additional further seven days. Cells were stained with CD138-PE and anti-human IgD-FITC. Fig. 11 A is a pair of scatter plots of the population of CDl 38⁺ plasma cells. Dark lines show the gating for cells analyzed for intracellular IgG expression quantitated as shown in Figs. 11B and 11C.

Fig. 1 IB is a bar graph depicting the results of a flow cytometry analysis in which cells expressing CDl 38 after incubation in the presence or absence of ferritin (cell events gated) were stained intracellularly with anti-IgG to identify plasma cells (CD138⁺ cells) expressing IgG.

Fig. 11C is a bar graph depicting the results of a flow cytometry analysis in which cells expressing CDl 38 (cell events gated) after incubation in the presence of supernatant prepared from cells expressing nef (Ad-nef), GFP (Ad-GFP) or mock infected were stained with anti-IgG intracellularly to identify plasma cells (CD138⁺ cells) expressing IgG.

Fig. 12 A is a graph illustrating the results of experiments in which peripheral blood lymphocytes were incubated in H-thymidine, ferritin, and anti-IgD (17.5 μg/ml).

Fig. 12B is a graph illustrating the results of experiments in which peripheral blood lymphocytes were incubated in ³H-thymidine, ferritin, and tetanus toxoid (1.3 μg/ml).

Fig. 12C is a graph illustrating the results of experiments in which peripheral blood lymphocytes were incubated in ³H-thymidine, ferritin, and phytohemagglutinin P (PHA-P; 1 μg/ml).

Fig. 13A is a graph illustrating the results of experiments in which B cells were incubated in ³H-thymidine, ferritin, and tetanus toxoid, anti-IgD, or "irrelevant protein" (Bovine Serum Albumin; BSA). Fig. 13B is a bar graph depicting the results of experiments in which B cells were incubated in normal medium or in medium containing anti-IgD (25 μg/ml), rCD40L (50 ng/ml), and IL-4 (10 ng/ml).

Fig. 14 is a graph depicting the results of experiments in which RAMOS B cells were incubated in increasing concentrations of ferritin for four days, washed, incubated for an additional five days. The amount of IgG secretion was then measured using ELISA (enzyme-linked immunosorbant assay). DETAILED DESCRIPTION

It has been discovered that ferritin, a protein secreted by HIN-1 infected primary macrophages, can induce known manifestations of the B lymphocyte dysfunction observed during the course of human HIN infection. The invention is based on newly discovered properties of ferritin: it can cause B cell hyperactivation, proliferation, and maturation; hypergammaglobulinaemia; and inhibition of an antigen-specific B cell response. The correlation between serum ferritin and viral load is confirmed (Example 1) as well as a correlation between ferritin and the degree of hypergammaglobulinaemia in HlY-infected patients (Example 1). Ferritin promotes B lymphocyte proliferation and induces expression of certain immunoglobulin isotypes (Example 2). Ferritin enhanced the growth of B cell lymphomas, which are the second most common cancer to occur during HIV infection (Example 3). The HIV-1 gene product that is responsible for all of these effects has been identified as the accessory protein Νef (Examples 4 and 5). It is also demonstrated herein that previously proposed mediators of B cell abnormalities in HIV infection are not involved in mediating such B cell abnormalities (Examples 4 and 5). This is demonstrated in that presence of proposed mediators is not required to recreate B cell dysfunction (for example, gpl20, IL-10) or that their presence has little, if any, effect upon these parameters of B cell function (for example, IL-6). However, purified human liver ferritin, recombinant light chain ferritin, ferri tin-containing supematants from HIV-1 infected macrophage cultures, and ferritin-containing supematants from macrophages transduced with only the HIV- 1 gene nef all possessed this potent B lymphocyte immunomodulatory activity. HIV- 1 Νef is essential for viral pathogenicity in vivo. Its functions include binding to CD4, and downregulation of cell surface CD4 expression, enhancement of HIV-1 replication in primary T cells, and binding to several cellular protein kinases, including serine/threonine kinases and protein tyrosine kinases (PTKs).

It has also been found that HIV-1 -infected macrophages or macrophages expressing the accessory protein Νef, produce a factor that promotes B cell proliferation and differentiation to immunoglobulin-secreting plasma cells. Infected macrophages and Νef-expressing macrophages released elevated levels of homopolymeric light (L)-chain ferritin. Inhibition of ΝFKB induction by Νef reduced ferritin release despite Nef acting posttranscriptionally on ferritin mRNA. The induction of B cell proliferation and differentiation by Nef- expressing macrophages was blocked after immunodepletion of ferritin while addition of ferritin reconstituted these activities. Ferritin initiated signals in B cells leading to expression of genes that can regulate cell-cycle progression, protection from apoptosis and immunoglobulin production. It was also observed that there is a significant correlation between plasma viral load, ferritin concentration, and serum immunoglobulin in HIV-1 infected individuals. Therefore, ferritin has activities that extend beyond its role in iron metabolism and the induction of ferritin from infected macrophages is related to the mechanism by which viral replication promotes collateral B cell defects in HIV-1 - infected individuals.

The invention relates to methods of inducing B cell proliferation, either in a subject or in culture by contacting a B cell with ferritin or a compound that increases ferritin expression or activity. In the case of induction of B cell proliferation in culture, the cells can be genetically engineered B cells (e.g., to produce a recombinant antibody). Methods of producing such cells are known in the art. After induction of proliferation, and, in general, subsequent maturation (e.g., expression of antibody), the cells can be introduced into a subject, thereby providing the subject with B cells. One advantage of this method is that B cells can be obtained from a subject that is deficient in B cells, induce proliferation as described herein, and introduce the expanded B cell population into the subject, thus providing them with autologous B cells.

The invention also encompasses methods of treating HIV infection by inhibiting ferritin expression or activity. Thus, ferritin can be used as a target for therapeutic interventions for HIV infections. Ferritin can also be used as a therapeutic agent for induction of B lymphocyte proliferation. Ferritin levels can be monitored to assess or correlate a degree of B lymphocyte dysfunction. This diagnostic use is novel in that high serum ferritin levels in patients have only been reported to correlate with a high viral load and a poor prognosis. Antisense Nucleic Acid Molecules, Ribozymes and Modified Ferritin Nucleic Acid Molecules

In some embodiments, the invention includes isolated nucleic acid molecules that are antisense to ferritin nucleic acids. Such molecules are useful for modulating the expression of ferritin. An "antisense" nucleic acid can include a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire ferritin coding strand, or to only a portion thereof (e.g., the coding region of human ferritin corresponding to, for example, Genbank ^®

Accession Nos. P02792, NP000137, and M1019. Genbank^® accession numbers for ferritin light chain are, e.g., NM000146, BU074189 and BU074966 and examples of ferritin heavy chain are, e.g., BU075632, BU076814, XM171637, XM042852, LOC167011 and LOC220667. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding ferritin (e.g., the 5' and 3' untranslated regions).

An antisense nucleic acid can be designed such that it is complementary to the entire coding region of ferritin mRNA, but is generally an oligonucleotide which is antisense to only a portion of the coding or noncoding region of ferritin mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of ferritin mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length. Antisense nucleic acids can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art and commercial services are available for design and synthesis (e.g., Molecula Research Labs, Herndon, VA). For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules used in the new methods are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a ferritin protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using vectors described herein and vectors known in the art. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred. In some embodiments, the antisense nucleic acid molecule is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analog (Inoue et al. 1987, FEBS Lett. 215:327-330).

In still another embodiment, an antisense nucleic acid is a ribozyme. A ribozyme having specificity for a ferritin-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a ferritin cDNA, and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach 1988, Nature 334:585-591; Turner, 1997, Ribozyme Design, Humana Press). For example, a derivative of a Tetrahymena L-19 INS RΝA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a feπϊtin-encoding mRΝA. See, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, ferritin mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., 1993, Science 261 :1411-1418.

Ferritin gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a ferritin gene (e.g., the ferritin promoter and/or enhancers) to form triple helical structures that prevent transcription of the ferritin gene in target cells (e.g., Helene, 1991, Anticancer Drug Des. 6:569-84;

Helene, 1992, Ann. N.Y. Acad. Sci. 660:27-36; and Maher, 1992, Bioassays 14:807- 15). The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

The invention also provides detectably labeled oligonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or colorimetric.

A ferritin nucleic acid molecule can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, e.g., the stability (e.g., to increase half-life and expression of ferritin), hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup et al., 1996, Bioorganic & Medicinal

Chemistry 4:5-23). As used herein, the terms "peptide nucleic acid" or "PNA" refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone, and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996, supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs of ferritin nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of ferritin nucleic acid molecules can also be used as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup et al., 1996, supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup et al., 1996, supra; Perry-O'Keefe, supra).

In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648- 652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, Bio-Techniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

The invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to a ferritin nucleic acid, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence of the ferritin nucleic acid in a sample. Such uses include monitoring ferritin expression in screening assays to identify compounds that modulation ferritin expression. Molecular beacon nucleic acids are described, for example, in Lizardi et al. (U.S. Patent No. 5,854,033), Nazarenko et al. (U.S. Patent No. 5,866,336), and Livak et al. (U.S. Patent 5,876,930). Recombinant Expression Vectors, Host Cells and Genetically Engineered Cells

Some embodiments include vectors, generally expression vectors, containing a nucleic acid encoding a polypeptide described herein. Such vectors can be used to provide ferritin to a cell, e.g., to increase B cell proliferation and maturation. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid, or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retro viruses, adenoviruses, and adeno-associated viruses. Recombinant expression vectors are useful, e.g., for introducing sequences into cells that code for antisense RNAs or ribozymes that decrease ferritin expression. Recombinant expression vectors are also useful for introducing nucleic acid sequences into a cell that either code for ferritin or for factors that can induce ferritin expression. Such expression is useful for inducing proliferation of B lymphocytes. A vector can include a ferritin nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., ferritin proteins, mutant forms of ferritin proteins, fusion proteins, and the like).

The recombinant expression vectors can be designed for expression of ferritin proteins in prokaryotic or eukaryotic cells. For example, polypeptides can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990, Gene Expression Technology: Methods in Enzvmolo V 185, Academic Press, San Diego, CA). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non- fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S- transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Purified fusion proteins can be used in ferritin activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for ferritin polypeptides using methods known in the art. In one embodiment, a fusion protein expressed in a retroviral expression vector of the present invention can be used to infect bone marrow cells that are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six weeks).

To maximize recombinant protein expression in E. coli is to express the protein in a host bacterium with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, 1990, Gene Expression Technology: Methods in Enzymology_185, Academic Press, San Diego, California, pp. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., 1992, Nucleic Acids Res. 20:2111- 2118). Such alteration of nucleic acid sequences can be carried out by known DNA synthesis techniques.

The ferritin expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector, or a vector suitable for expression in mammalian cells.

When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., 1987, Genes Dev. 1 :268-277), lymphoid- specific promoters (Calame and Eaton, 1988, Adv. Immunol. 43:235-275), promoters of T cell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) and immunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen and Baltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al., 1985, Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss, 1990, Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989, Genes Dev. 3:537-546). B lymphocyte-specific promoters can be derived naturally or synthetically constructed. These promoters can include the continually active promoter flanking the B cell protein, EBF, (Smith et al., 2002, J. Immunol. 169:261- 70), or DNA elements containing binding sites for octamer factors and the co- activator OBF that are active at multiple stages of B cell development (Laumen et al., 2000, Eur. J. Immunol. 30:458-69). Promoters from individual B cell differentiation antigens may be employed to direct expression at specific points in the cells maturation, such as CDl 9 (Riva et al., 1997, J. Immunol. 159:1284-92, or CD20 (Riley et al., 2000, Sem. Oncol. 27(6 Suppl. 12): 17-24).

In other embodiments a recombinant expression vector is used comprising a DNA molecule cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus. For a discussion of the regulation of gene expression using antisense genes (e.g., Weintraub et al., 1986, Trends in Genetics 1 :1).

Another aspect the invention provides a host cell that includes a nucleic acid molecule described herein, e.g., a ferritin nucleic acid molecule within a recombinant expression vector or a ferritin nucleic acid molecule containing sequences that allow it to homologously recombine into a specific site of the host cell's genome. The terms "host cell" and "recombinant host cell" are used interchangeably herein. Such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, a ferritin protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DΕAΕ-dextran-mediated transfection, lipofection, or electroporation. A cell can produce (i.e., express) a ferritin protein. Accordingly, methods are provided for producing a ferritin protein in a cell, e.g., by a B lymphocyte. In one embodiment, the method includes culturing the host cell (into which a recombinant expression vector encoding a ferritin protein has been introduced) in a suitable medium such that a ferritin protein is produced. In another embodiment, the method further includes isolating a ferritin protein from the medium or the host cell. The cell can then be used, e.g., for transplantation into a subject.

In another aspect, the invention features a cell or purified preparation of cells that include a ferritin transgene, or which otherwise misexpress ferritin. The cell preparation can consist of human or non-human cells, e.g., non-human primate, rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In some embodiments, the cell or cells include a ferritin transgene, e.g., a heterologous form of a ferritin, e.g., a gene derived from humans (in the case of a non-human cell). The ferritin transgene can be misexpressed, e.g., overexpressed or underexpressed with respect to a control such as a wild type cell (for example, a cell of the same type from an individual that is not HIV infected). In other embodiments, the cell or cells include a gene that mis- expresses an endogenous ferritin, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders (e.g. HIN) that are related to mutated or mis-expressed ferritin alleles or for use in drug screening.

In another aspect, the invention features, a human cell, e.g., a hematopoietic stem cell, transformed with nucleic acid that encodes a ferritin polypeptide.

Also provided are cells, e.g., human hematopoietic or fibroblast cells, in which an endogenous ferritin gene is under the control of a regulatory sequence that does not normally control the expression of the endogenous ferritin gene. The expression characteristics of an endogenous gene within a cell, e.g., a cell line or microorganism, can be modified by inserting a heterologous DΝA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the endogenous ferritin gene. For example, an endogenous ferritin gene that is "transcriptionally silent," e.g., not normally expressed, or expressed only at very low levels, can be activated by inserting a regulatory element that can promote the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombination can be used to insert the heterologous DNA as described in, e.g., Chappel (U.S. patent no. 5,272,071 and WO 91/06667).

Ferritin as used herein includes the H subunit and the L subunit of ferritin. The ferritin used, e.g., to induce B cell proliferation and maturation can be composed of a mixture of subunits or of only one subunit (e.g., the L subunit). Ferritin proteins are known in the art. Any ferritin or portion of a ferritin polypeptide can be used in the methods described herein as long as it is able to exhibit the desired activity, e.g., the ability to induce B lymphocyte proliferation and maturation. Thus, this activity provides a test for identification of an active fragment of a ferritin polypeptide. Ferritin can be used from the same or a different species from a cell that is used in a method described herein. The ferritin is regarded as suitable for use in the methods by its ability to exhibit a ferritin activity, e.g., induction of proliferation of the cell.

Prevention ofB Lymphocyte Proliferation The invention encompasses methods of screening for compounds that prevent

B lymphocyte proliferation and the use of such compounds, e.g., to treat disorders associated with B lymphocyte proliferation. Compounds useful in the invention are compounds that decrease or prevent the induction of B cell proliferation by ferritin. Screening assays such as those described herein can be used to identify candidate compounds. Such compounds include those that inhibit one or more of the effects of ferritin including proliferation of B lymphocytes, expression of antibody isotypes, and the ability of ferritin to suppress antigen-specific immune responses, i.e., to act in an immunosuppressive manner. In Figs. 12 and 13, ferritin is shown to suppress the early (proliferative) response of leukocytes to the common recall antigen tetanus toxoid. Therefore, it can form the basis of modified compounds that possess a stronger effect in this regard for the purposes of eliciting immunosuppression in, for example, organ transplant patients.

Induction ofB Lymphocyte Proliferation In certain cases, induction ofB lymphocyte proliferation is desirable. Such proliferation can be induced in vitro or in vivo. Examples of circumstances under which induction is desirable include stimulation ofB cell proliferation in cells removed from subjects for ex vivo procedures. Ferritin can be used to expand B cells isolated from a patient and transduced with vectors to express specific transgenes such as genetically engineered recombined antibody (Ig) 'mini-genes' of a desired known specificity, or to promote an augmented antibody response following vaccination to a specific disease agent or cancer-specific antigen. For example, the ex vivo expansion ofB lymphocytes increases the efficiency of current gene delivery techniques since engineered cells expressing the desired protein can be selected in culture, then expanded. This permits the delivery of functional copies of defective genes such as BCL 2 or BCL 6, which are associated with the occurrence ofB cell lymphoma (Muramatsu et al., 1996, Br. J. Haematol. 93:911-20). Ferritin can also be used to augment an antibody response to vaccine preparations by ex vivo or in vivo methods detailed below. In some embodiments, ex vivo expansion ofB cells for the purpose of ferritin acting as a vaccine adjuvant requires the removal or dilution of exogenously added ferritin. The B cell differentiates to a plasma cell secreting antibodies to the subsequently added immunogen without the immunosuppressive effects of ferritin reported herein. Ferritin-induced B cell proliferation can augment immune responses to vaccine preparations by enhancing the antibody response through ferritin-driven B cell proliferation and differentiation when incorporated in the vaccination with the immunogen at a ferritin dose below that determined to be immunosuppressive. Induction can be performed by direct addition of ferritin or forms of ferritin that can induce B lymphocyte proliferation. This includes ferritin light chain, ferritin polypeptides shown to induce proliferation in test systems such as those in the Examples below.

Screen ing A ssays

The invention includes the identification of compounds that can inhibit ferritin expression or activity. Compounds that affect ferritin expression (nucleic acid or protein) or activity are useful, e.g., for modulating B lymphocyte maturation. Assays for such compounds can be performed using the methods described below. The invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) that bind to ferritin proteins, have a stimulatory or inhibitory effect on ferritin expression or ferritin activity. Compounds thus identified can be used to modulate the expression or activity of ferritin in a therapeutic protocol e.g., to modulate B lymphocyte maturation. In one embodiment, the assays are for screening candidate or test compounds that are substrates of a ferritin protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate the activity of a ferritin protein or polypeptide or a biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al., 1994, J. Med. Chem. 37:2678-2685); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one- bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12: 145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91 :11422; Zuckermann et al, 1994, J. Med. Chem. 37:2678; Cho et al., 1993, Science 261 :1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al., 1994, J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science 249:386- 390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner supra.).

In one embodiment, an assay is a cell-based assay in that a cell expressing a ferritin protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate ferritin activity is determined. Determining the ability of the test compound to modulate ferritin activity can be accomplished by monitoring, for example, B lymphocyte proliferation in the presence of ferritin and in the presence and absence of the test compound. The cell, for example, can be of mammalian origin, e.g., human. Methods of detecting cell proliferation are known in the art (e.g., using BrdU incorporation or ³thymidine incorporation) and B cells can specifically be selected before use in the assays (e.g., by panning using B-cell specific antibodies) or by detecting cells using B-cell specific markers. Some such methods are described in the Examples.

The ability of the test compound to modulate ferritin binding to a compound, e.g., a ferritin substrate can be determined. For example, live cells labeled with fluorescently labeled ferritin have been used to detect the presence of ferritin binding sites by flow cytometry. Compounds that interfere with ferritin binding can be identified using this method. For example, a test compound is incubated with cells and labeled ferritin. The ability of the test compound to reduce ferritin binding to the cells (e.g., compared to a control incubation mixture that does not contain the test compound) is then measured. Test compounds that interfere with ferritin binding reduce the amount of ferritin binding to the cell. With modification, such an assay can also be performed using other ferritin-containing substrates, e.g., isolated plasma membranes or fixed cells that are cyto-spun onto a solid support. H-kininogen, present in human serum is a ferritin-binding protein and can be used in such assays (Torti et al. 1998, J. Biol. Chem. 273:13630-136305). In another assay method, the ability of a test compound to bind to ferritin is evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, to a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to ferritin can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, ferritin could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate ferritin binding to a ferritin substrate in a complex. For example, compounds (e.g., ferritin substrates) can be labeled with 125j₅ 35g_} IAQ_{^ or} 3fj, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

The ability of a compound (e.g., a ferritin substrate) to interact with ferritin, with or without the labeling of any of the interactants, can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with ferritin without the labeling of either the compound or the ferritin (see, McConnell et al., 1992, Science 257:1906-1912). As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and ferritin.

In yet another embodiment, a cell-free assay is provided in which a ferritin protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the ferritin protein or biologically active portion thereof is evaluated. In general, biologically active portions of the ferritin proteins to be used in assays of the present invention include fragments that participate in interactions with non-ferritin molecules, e.g., fragments with high surface probability scores.

Soluble and/or membrane-bound forms of isolated proteins (e.g., ferritin proteins or biologically active portions thereof) can be used in the cell-free assays. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N- methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-l 14, Thesit®, Isotridecypoly (ethylene glycol ether)_n, 3-[(3- cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylamminio]-2-hydroxy-l -propane sulfonate (CHAPSO), or N-dodecyl=N, N-dimethyl-3-ammonio-l -propane sulfonate.

Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Patent No. 5,631,169; Stavrianopoulos et al, U.S. Patent No. 4,868,103). A fluorophore label on the first, 'donor' molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor.' Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

In another embodiment, determining the ability of the ferritin protein to bind to a target molecule can be accomplished using real-time Biomolecular interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal, which can be used as an indication of real-time reactions between biological molecules. In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Generally, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

To facilitate separation of complex ed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay either ferritin, an anti-ferritin antibody or its target molecule can be immobilized. Binding of a test compound to a ferritin protein, or interaction of a ferritin protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/ferritin fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione Sepharose™ beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or ferritin protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of ferritin binding or activity determined using standard techniques.

Other techniques for immobilizing either a ferritin protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated ferritin protein or target molecules can be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce

Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactive with ferritin protein or target molecules but which do not interfere with binding of the ferritin protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or ferritin protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the ferritin protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the ferritin protein or target molecule.

Alternatively, cell-free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas and Minton, 1993, Trends Biochem. Sci. 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, 1998, J. Mol. Recognit. 11 :141-8; Hage and Tweed, 1997, J.

Chromatogr. B. Biomed. Sci. Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

In one embodiment, the assay includes contacting the ferritin protein or biologically active portion thereof with a known compound that binds ferritin to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a ferritin protein, wherein determining the ability of the test compound to interact with a ferritin protein includes determining the ability of the test compound to preferentially bind to ferritin or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

The target gene products can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as "binding partners." Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The target genes/products for use in this embodiment are generally the ferritin genes/products herein identified, however others known in the art can be used. In an alternative embodiment, the invention provides methods for determining the ability of the test compound to modulate the activity of a ferritin protein through modulation of the activity of a downstream effector of a ferritin target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described. To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below. In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

In an alternate embodiment, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

In yet another aspect, the ferritin proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al., 1993, Cell 72:223-232; Madura et al., 1993, J. Biol. Chem. 268:12046-12054; Bartel et al., 1993, Biotechniques 14:920-924; Iwabuchi et al., 1993, Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with ferritin ("ferritin-binding proteins" or "ferritin-bp") and are involved in ferritin activity. Such ferritin-bps can be activators or inhibitors of signals by the ferritin proteins or ferritin targets as, for example, downstream elements of a ferritin- mediated signaling pathway.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a ferritin protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. Alternatively the: ferritin protein can be the fused to the activator domain. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a ferritin- dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., lacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein that interacts with the ferritin protein. In another embodiment, modulators of ferritin expression are identified. For example, a cell or cell-free expression system mixture is contacted with a candidate compound and the expression of ferritin mRNA or protein evaluated relative to the level of expression of ferritin mRNA or protein in the absence of the candidate compound. When expression of ferritin mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of ferritin mRNA or protein expression. Alternatively, when expression of ferritin mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of ferritin mRNA or protein expression. The level of ferritin mRNA or protein expression can be determined by methods described herein for detecting ferritin mRNA or protein. In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a ferritin protein can be confirmed in vivo, e.g., in an animal such as a non-human primate model for HIV infection.

This invention further pertains to novel agents identified by the above- described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a ferritin modulating agent, an antisense ferritin nucleic acid molecule, a ferritin-specific antibody, or a ferritin- binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

Pharmaceutical Compositions Compounds, including nucleic acids, polypeptides, fragments thereof, and anti-ferritin antibodies (also referred to herein as "active compounds") can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal, and rectal administration. Compounds can also be administered orally. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic admimstration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, about 0.01 to 25 mg/kg body weight, about 0.1 to 20 mg/kg body weight, or about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

In general, a dosage of ferritin is provided such that serum ferritin levels in the subject are between about at least about 13 ng/ml, at least about 20 ng/ml, at least about 50 ng/ml, at least about 100 ng/ml, or higher. In general, the desirable range is from about 30-300 ng/ml and it is generally undesirable that levels be greater than 400 ng/ml (see, e.g., Merck Manual, 16^th edition, Merck & Co., Inc., Rahway, N.J.). Serum ferritin can be assayed using methods known in the art, including radioimmunoassay (RIA).

For antibodies, the dosage is generally 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. (1997, J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

The present invention encompasses agents that modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated. An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates described herein can be used for modifying a given biological response such as B lymphocyte proliferation; the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("LL- 1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.

The nucleic acid molecules described herein can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al., 1994, Proc. Natl. Acad. Sci. USA 91 :3054-3057). The pharmaceutical preparation of the gene therapy vector can - include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted ferritin expression or activity. As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

In one aspect, a method is provided for preventing in a subject, a disease or condition associated with an aberrant or unwanted ferritin expression or activity, by administering to the subject ferritin or an agent that modulates ferritin expression or at least one ferritin activity. Subjects at risk for a disease that is caused or contributed to by aberrant or unwanted ferritin expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the ferritin aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of ferritin aberrance, for example, ferritin or a ferritin agonist can be used to stimulate B lymphocyte proliferation. A ferritin antagonist agent can be used for treating a subject with suppressed B lymphocyte proliferation, e.g., a subject infected with HIV. The appropriate agent can be determined based on screening assays described herein. Ferritin can act as a novel diagnostic target for disorders related to B lymphocyte proliferation and therapeutic agents for controlling such disorders including HIV infection.

As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. "Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair. Pathologic proliferative disorders that can be treated include lymphoid malignancies and include, but are not limited to acute lymphoblastic leukemia (ALL), which includes B-lineage ALL.

The ferritin nucleic acids and proteins and compounds that modulate ferritin expression or activity can be used to treat and or diagnose a variety of immune disorders. The ability of ferritin to ameliorate the symptoms or progression of immunopathological diseases is generally related to the degree to which the humoral (antibody) arm of immunity is involved. For example, antibody-mediated autoimmune conditions can be suppressed using ferritin. The antigen-specific immunosuppressive effect reported in herein may also act at the level of antigen presentation and thus impairs B cell dependent cytotoxic T cell responses to thymus dependent antigens - thymus dependent antigens require T cells for immune recognition. This type of immune response is also deficient in HIV infected patients. T cell dependent immunity or rather the loss of it occurring through CD4+ T cell depletion - the hallmark of HIV infection and causes ADDS. Examples of disorders or diseases that can be treated or diagnosed using methods described herein include, but are not limited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjόgren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens- Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft- versus-host disease, cases of transplantation, and allergy such as, atopic allergy.

As discussed, successful treatment ofB lymphocyte related disorders can be achieved using techniques that inhibit the expression or activity of ferritin gene products. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of HIV disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab')₂ and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

Further, antisense and ribozyme molecules that inhibit expression of a ferritin gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Normal ferritin protein can be administered such that it enters a cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

Aptamer molecules specific for ferritin can also be used to treat B lymphocyte-related disorders. Aptamers are nucleic acid molecules having a tertiary structure that permits them to specifically bind to protein ligands (see, e.g., Osborne et al., 1997, Curr. Opin. Chem. Biol. 1 :5-9; and Patel, 1997, Curr. Opin. Chem. Biol. 1 :32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which ferritin protein activity can be specifically decreased without the introduction of drugs or other molecules that may have pluripotent effects.

Antibodies can be generated that are both specific for ferritin and that reduce ferritin activity. Such antibodies may, therefore, by administered in instances where negative modulatory techniques are appropriate for the treatment of B lymphocyte- related disorders. Antibodies that specifically bind to ferritin or fragments thereof are known in the art and can be made using known methods such as those described herein. In circumstances wherein injection of an animal or a human subject with a ferritin protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against ferritin through the use of anti-idiotypic antibodies (see, for example, Herlyn, 1999, Ann. Med. 31 :66-78; and Bhattacharya-Chatterjee and Foon, 1998, Cancer Treat. Res. 94:51-68). If an anti- idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the ferritin protein. Vaccines directed to a disease characterized by ferritin expression can also be generated in this fashion.

In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies are generally used. Lipofectin^® or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al., 1993, Proc. Natl. Acad. Sci. USA 90:7889-7893).

The identified compounds that inhibit ferritin gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate B lymphocyte-related disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above. In the case where proliferation ofB lymphocytes is undesirable, an effective amount or dose of a compound is one that decreases the number ofB lymphocytes or proliferation ofB lymphocytes in a subject relative to a reference such as the subjects B lymphocyte titer prior to treatment.

Data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. Another example of determination of effective dose for an individual is the ability to directly assay levels of "free" and "bound" compound in the serum of the test subject. Such assays may utilize antibody mimics and/or "biosensors" that have been created through molecular imprinting techniques. The compound which is able to modulate ferritin activity is used as a template, or "imprinting molecule", to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix that contains a repeated "negative image" of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell et al. (1996, Current Opinion in Biotechnology 7:89-94) and in Shea (1994, Trends in Polymer Science 2:166-173). Such "imprinted" affinity matrixes are amenable to ligand-binding assays in which an immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way is reported in Vlatakis et al. (1993, Nature 361 :645-647). Through the use of isotope labeling, the "free" concentration of compound that modulates the expression or activity of ferritin can be readily monitored and used in calculations of IC₅₀. Such "imprinted" affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiber optic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC ₀. A rudimentary example of such a "biosensor" is discussed in Kriz et al. (1995, Analytical Chemistry 67:2142-2144).

Methods of modulating ferritin expression or activity for therapeutic purposes can also be practiced. Accordingly, in an exemplary embodiment, a modulatory method involves contacting a cell with a ferritin or agent that modulates one or more of the activities of ferritin protein activity associated with the cell. An agent that modulates ferritin protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a ferritin protein (e.g., a ferritin substrate or receptor), a ferritin antibody, a ferritin agonist or antagonist, a peptidomimetic of a ferritin agonist or antagonist, or other small molecule. In one embodiment, the agent stimulates one or ferritin activities. Examples of such stimulatory agents include active ferritin protein and a nucleic acid molecule encoding ferritin. In another embodiment, the agent inhibits one or more ferritin activities. Examples of such inhibitory agents include antisense ferritin nucleic acid molecules, antiferritin antibodies, and ferritin inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a ferritin protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up regulates or down regulates) ferritin expression or activity. In another embodiment, the method involves administering a ferritin protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted ferritin expression or activity.

Stimulation of ferritin activity is desirable in situations in which ferritin is abnormally downregulated and/or in which increased ferritin activity is likely to have a beneficial effect. For example, stimulation of ferritin activity is desirable in situations in which a ferritin is downregulated and/or in which increased ferritin activity is likely to have a beneficial effect. Likewise, inhibition of ferritin activity is desirable in situations in which ferritin is abnormally upregulated and/or in which decreased ferritin activity is likely to have a beneficial effect.

Predictive Medicine

The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual. In some embodiments, the invention includes assays related to the novel properties of ferritin on B cells. Such assays can be diagnostic, prognostic, or for monitoring ferritin levels, e.g., to monitor a disease such as HIV. Testing of HIV patient sera for ferritin can predict the degree ofB cell dysfunction due to hyperferritinemia and have prognostic value. For example, following determination of serum ferritin levels by ELISA, patient serum is immunodepleted with antiserum to ferritin and an isotype control antiserum as described infra. The serum is then tested for the ability to induce Ig secretion from the RAMOS B cell line, as described herein, compared to sera from uninfected patient controls. Since re-sterilization by filtration would ensure that the serum is completely cell free, the induction ofB cell hyperactivation (proliferation) or Ig secretion can be tested using primary B lymphocytes using methods known in the art such as those described herein.

The method includes detecting, in a tissue of the subject, the expression of a ferritin gene, at the mRNA level, e.g., detecting a level of ferritin expression that is associated with an adverse symptom of HIV infection; or detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a level of ferritin expression that is associated with an adverse symptom of HIV infection. Detection of ferritin expression or activity can be used to monitor the efficacy of a treatment regime for HIV, including those specifically designed to reduce ferritin levels in the subject.

In certain embodiments detecting the mis-expression includes ascertaining the existence of an alteration in the level of a messenger RNA transcript of a ferritin gene or a level of ferritin different from that associated with healthy (non-HIV infected) subjects.

Methods that identify the abnormal expression or activity of ferritin can be used prenatally or to determine if a subject's offspring will be at risk for a disorder. In some embodiments the method includes contacting a sample from a subject with an antibody to the ferritin protein or a nucleic acid, which hybridizes specifically with the ferritin gene. These and other embodiments are discussed below.

Diagnostic and Prognostic Assays

The presence, level, or absence of ferritin protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent that can detect ferritin protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes ferritin protein such that the presence of ferritin protein or nucleic acid is detected in the biological sample. Such methods are useful, e.g., for detecting levels in a subject in which induction or suppression ofB lymphocyte proliferation is desired. For example, in a subject with a proliferative disorder related to overproduction ofB lymphocytes, assaying ferritin levels is useful to determine whether they are elevated and thus administering a compound that inhibits ferritin expression or activity is a possible treatment. Furthermore, assays of ferritin are useful for monitoring the efficacy of treatments to decrease ferritin levels in treating, e.g., a proliferative disorder related to B-lymphocytes.

The term "biological sample" includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. In general, the biological sample is serum. The level of expression of a ferritin gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the ferritin genes; measuring the amount of protein encoded by the ferritin genes; or measuring the activity of the protein encoded by the ferritin genes. The level of mRNA corresponding to the ferritin gene in a cell can be determined both by in situ and by in vitro formats.

The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length ferritin nucleic acid, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to ferritin mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays are described herein.

In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the ferritin genes.

The level of mRNA in a sample that is encoded by one of ferritin can be evaluated with nucleic acid amplification, e.g., by RT-PCR (Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6: 1197), rolling circle replication (Lizardi et al., U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice- versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the ferritin gene being analyzed.

In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting ferritin mRNA, or genomic DNA, and comparing the presence of ferritin mRNA or genomic DNA in the control sample with the presence of ferritin mRNA or genomic DNA in the test sample.

A variety of methods can be used to determine the level of protein encoded by ferritin. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label.

Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein. The detection methods can be used to detect ferritin protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of ferritin protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of ferritin protein include introducing into a subject a labeled anti-ferritin antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting ferritin protein, and comparing the presence of ferritin protein in the control sample with the presence of ferritin protein in the test sample.

The invention also includes kits for detecting the presence of ferritin in a biological sample. For example, the kit can include a compound or agent capable of detecting ferritin protein or mRNA in a biological sample and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect ferritin protein or nucleic acid.

For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker (e.g., of a ferritin polypeptide); and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can include: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker (e.g., of a ferritin polypeptide) or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker (e.g., a nucleic acid sequence encoding a ferritin polypeptide or untranslated region of such a molecule). The kit can also includes a buffering agent, a preservative, or a protein stabilizing agent. The kit can also includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples that can be assayed and compared to the test sample contained. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or aberrant or unwanted ferritin expression or activity. As used herein, the term "unwanted" includes an unwanted phenomenon involved in a biological response such as pain or deregulated cell proliferation.

In one embodiment, a disease or disorder associated with aberrant or unwanted ferritin expression or activity is identified. A test sample is obtained from a subject and ferritin protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level, e.g., the presence or absence, of ferritin protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted ferritin expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g., serum), cell sample, or tissue.

The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with abenant or unwanted ferritin expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for that prevents B lymphocyte proliferation.

The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose or monitor patients exhibiting symptoms of, e.g., a disease involving proliferation ofB lymphocytes.

EXAMPLES

Example 1 : Induction ofB Lymphocytes, but not T Lymphocyte, Proliferation by Supematants from Macrophages Expressing HIV-1 Nef

Supematants prepared from macrophages expressing HIV-1 nef protem were tested for the presence of a soluble factor that affects lymphocyte proliferation. In these experiments macrophages were transduced with adenovirus vector containing an HIV nef gene, an irrelevant control, the green fluorescent protein gene (GFP), or no adenovirus (mock). The vector was synthesized and used as described in Swingler et al. 1999, Nat. Med. 5:997-1003. Macrophages were prepared for transduction as described previously (Swingler et al., 1999, supra). Elutriated lymphocytes were purified into populations of T or B lymphocytes by negative selection with antibody coupled paramagnetic beads according to manufacturers' instructions (Dynal Biotech, Oslo, Norway). Purity was determined to be greater than 96% by flow cytometric staining using fluorochrome antibodies to CD3 (T cell), CD 19 (B cell), and CD45 (common leukocyte) (BD Pharmingen, San Diego, CA). Cells were cultured at 5 million per ml in RPMI-1640 medium containing 5% fetal bovine serum. Cultures were incubated in a 1 :3 dilution of supernatant derived from 700,000 macrophages transduced with recombinant adenovirus expressing HIV-1 Nef or in supernatant from macrophages that did not express HIV-1 Nef but expressed GFP, or had been mock infected. Incubation in the presence of anti-IgD (25 μg/ml) + rCD40L (recombinant human CD40L; 50 ng/ml ) + IL-4 (10 ng/ml) was used as a positive control for induction ofB cell proliferation. Tritiated thymidine was added to the cultures four days after the initiation of incubations (1 μCi) and the cultures were incubated for an additional 16 hours. Cells were then harvested, washed, and the amount of ³H- thymidine per 10⁶ cells was determined.

It was found that resting, primary B lymphocytes, but not T lymphocytes, entered the cell cycle in response to a soluble factor that was present in the cell culture supernatant of macrophages expressing HIV-1 ne protein (Figs. 1A and IB). Supematants from mock-infected and GFP-1 expressing cells did not induce B lymphocyte proliferation.

The induction of different immunoglobulins was also assessed in these experiments using methods known in the art. It was found that a soluble factor in the supernatant from HIV-1 nef expressing cells elicited high level production of different immunoglobulins compared to controls (Adeno-GFP and mock infected); isotypes IgA, IgG, and IgM (Table 1). Table 1

These results demonstrate that expression of HIV- 1 Nef in macrophages leads to the release of a soluble factor(s) that induces B cell proliferation, maturation, and differentiation to become antibody secreting cells. The molecular weight of the soluble factor was determined to be approximately 250 kilodaltons (kd) or greater. Unique proteins in this size range from supematants of macrophages expressing nef were subjected to micro-sequencing. Sequencing identified the presence of homopolymeric complexes of ferritin light chain (Figs. 2 A and 2B). The presence of ferritin was further confirmed with an ELISA assay analyzing the amount of ferritin released from adeno virus-transduced macrophages expressing Nef, GFP, or mock infected (Fig. 2C). Western blotting demonstrated the presence of ferritin in the concentrated supernatant from macrophages expressing Nef (Fig. 2D). These properties of ferritin are new and distinct from previously characterized factors that stimulate B lymphocytes. These data show that ferritin is associated with B lymphocyte proliferation and induction of immunoglobulin isotypes, two effects observed in individuals with HIV infection. These data also suggest that one method of decreasing ferritin release is to inhibit expression or activity of Nef. These data also demonstrate that ferritin can induce B lymphocyte proliferation and induce the expression of immunoglobulin isotypes.

Example 2: Secretion of Ferritin and Macrophage Colony Stimulating Factor (M- CSF) from HIV-1 Infected Macrophages

The secretion of ferritin and the effect of Nef expression in macrophages were investigated. The amounts of ferritin and M-CSF released into supematants of cultures infected with an HIV-1 wild type strain (ADA WT), HIV strain harboring a mutant Nef gene (ADA ΔNef), or mock infected were measured. Much less soluble ferritin was induced in cultures infected with an HIV-1 strain that had a mutant nef gene. There was also some slight variability between different donors. A low concentration of ferritin is also released by most cultures (Table 2). Repeated analyses confirmed the association between Nef expression and the induction of ferritin. Representative data are illustrated in Figs. 3A and 3B. These data demonstrate the dependence of ferritin production on expression of Nef. The HIN-1 induction of macrophage colony stimulating factor (M-CSF) was unaffected by mutation of the nef gene (Fig. 3C) as it was by transduction into macrophages of nef alone (Table 2).

Table 2

IgA, IgG, and IgM production was stimulated when B cells were exposed to supematants of macrophages replicating HIV-1 that harbor a normal nef gene.

Stimulation of immunoglobulin production was dependent on «e/(Figs. 4A, 4B, and 4C) and correlated with the increased concentrations of soluble ferritin in the supematants used in these experiments (Table 3).

Table 3

This phenomenon was not previously known. Thus, ferritin that is produced in response to HIV-1 ne/mediates a process analogous to the B lymphocyte hyperactivation and hypergammaglobulinaemia observed in human HIV-1 infection. To further substantiate the observation that ferritin is responsible for the immunoproliferative response ofB cells that is induced by HIV infection, macrophages were infected with adenovirus expression vectors containing a nef gene, a GFP gene, were mock infected, or were stimulated with rCD40L at 10 μg/ml. Supernatant from the infected cells was prepared, added to B lymphocyte cultures at a dilution of 1 :3, incubated for four days, and ³H-thymidine incorporation measured as described above. For some cultures, the supernatant was depleted for ferritin using an antibody that specifically binds ferritin. A non-specific isotype antibody was used as a depletion control. In these experiments, ferritin was removed by an immunoprecipitation technique. Rabbit polyclonal antiserum (US Biologicals, Swampscott, MA) against human ferritin and phosphate buffered saline washed

Protein A/G Sepharose™ (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the diluted macrophage supematants and incubated for 4 hours on a rotating platform at 4°C. Immune complexes were pelleted by centrifugation for 2 minutes at 750 x g. Supematants were re-sterilized by filtration through 0.22 micron syringe filters. These supematants were used in assays and compared to the results in which assays were performed using the same starting supernatant that was treated with an isotype control antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The titers of ferritin in the depleted supernatant were determined by ELISA assay (Table 4).

Table 4

The removal of ferritin from supematants of macrophages expressing «e/by immunodepletion almost completely abolished B cell activation (Fig. 5A). Nef- expressing macrophage supematants immunodepleted with an isotype antibody induced B cell proliferation.

In additional experiments examining IgM release from B cells after immunodepletion, it was observed that immunoglobulin production (IgM) was significantly less when cells were cultured in the presence of ferritin-depleted supernatant compared to incubation in control supematants (Fig. 5B and Table 5).

Table 5

These data show that ferritin plays a significant role in induction ofB lymphocyte proliferation and the induction of immunoglobulin production in HIV and that we/is responsible for induction of ferritin. Therefore, reduction of ferritin expression or activity, e.g., a reduction of ne induction of femtin is useful for ameliorating certain effects of HIV infection. These data also show that ferritin can be used to induce B lymphocyte proliferation and/or immunoglobulin production.

Example 3: Purified and Recombinant Ferritin Initiate Resting B Cell Progression into the Cell Cycle and Promote DNA Synthesis

To investigate the ability of ferritin to induce B lymphocyte proliferation, experiments were undertaken using purified ferritin and recombinant ferritin light chain. In these experiments, peripheral blood lymphocytes (PBLs) and B lymphocytes were isolated from healthy donors as follows. Lymphocytes obtained from peripheral blood by elutriation were used directly and are referred to as PBLs. Elutriated lymphocytes were purified into a population ofB lymphocytes by negative selection with antibody-coupled paramagnetic beads according to manufacturer's instructions (Dynal Biotech, Oslo, Norway). B cells were also purified by positive selection using antibody to the CD 19 cell surface antigen ofB cells coupled to paramagnetic particles following the manufacturer's instructions (Dynal Biotech, Oslo, Norway). Essentially identical results were obtained in these experiments using either method of purification. Cell purity was determined to be greater than 96% by flow cytometric staining using fluorochrome-labeled antibodies to CD3 (that identifies T cells), CD 19 (that identifies B cells), and CD45 (a common leukocyte marker) (BD Pharmingen, San Diego, CA). Cells were cultured at 5 million per ml in RPMI-1640 medium containing 5% fetal bovine serum, incubated in medium containing ferritin (from ICN, Costa Mesa, CA or Calbiochem, San Diego, CA) or ferritin light chain (Calbiochem, San Diego, CA) for four days. Tritiated thymidine was added to the cultures as described supra. After incubation, the cells were assayed for incorporation of H-thymidine.

Purified ferritin and recombinant ferritin light chain caused hyperactivation (proliferation) in peripheral blood lymphocytes (Figs. 6A and 6B) and purified B cells (Figs. 6C and 6D). In similar experiments, immunoglobulin isotypes were assayed. These experiments demonstrated that purified ferritin induced IgA, IgG, and IgM secretion from B lymphocytes (Figs. 7A, 7B, and 7C) as did recombinant ferritin. Fig. 7D shows the results of experiments detecting immunoglobulin secretion from B lymphocytes incubated in the presence anti-human IgD+IL-4+rCD40L, as a comparison. These data confirm that ferritin is the component of the supernatant that is inducing activation and induction of immunoglobulin secretion.

In contrast, immunoglobulin production was not induced after treatment ofB cells with recombinant IL-6 or MJP-lα at concentrations between 5 and 50,000 pg/ml. In the presence of ferritin L-chain, the frequency of CDl 38⁺ B cells was increased to a comparable extent as B cells incubated in the presence of a potent B cell differentiation stimulus. In these experiments, stimulation by ferritin was comparable to a proliferation and differentiation signal initiated by cross-linking the surface IgD signaling complex in the presence of IL-4 and CD40L.

The method described supra can be adapted to be used as an assay for compounds that decrease ferritin-induced B cell proliferation. For example, PBLs are incubated in the presence and absence of ferritin and in the presence and absence of a test compound. After incubation for sufficient time to permit detectable induction of proliferation in cells that are treated with ferritin but not the test compound, tritiated thymidine is added to the cultures. The cultures are incubated for sufficient time to permit incorporation of detectable amounts of tritiated thymidine into the cultures treated with ferritin. The amount of tritiated thymidine into the cultured cells is determined and the amount of tritiated thymidine incorporated into the different cultures is compared. Compounds that decrease amount of proliferation (as measured by ³H-thymidine incorporation) in cultures treated with ferritin compared to cultures that are treated with ferritin alone are candidate compounds for decreasing B lymphocyte proliferation.

Ferritin promoted the expression of kinase genes central to the transduction of mitogenic signals (e.g., MEK1/2, ERK2, the p38 kinases MKK3/6 and p38MAPK). MAPK pathways and the observed activation of NFKB (Fig. 5c) also favor the expression of anti-apoptotic and cell cycle promoting genes. Ferritin-stimulated B lymphocytes showed increased expression (>1.7x; of genes involved in suppression of apoptosis, in cell cycle progression, or in both processes (e.g., BCL-2, Caspcr/c-FLlP, cyclin D l , and NF-κB). Similarly, genes involved in the inhibition of cell cycle progression (e.g. p27^{κι ι} and p53) showed a significant down-regulation. This expression profile was comparable to the profile in B cells subjected to a proliferation differentiation stimulus (αIgD/IL-4/CD40L). In general, B cells from HIV-1 -infected individuals exhibit an aberrant proliferative response to a broad range ofB cell mitogens despite maintaining the ability to secrete immunoglobulin. These data support the finding that ferritin can be used in protocols to stimulate B cell proliferation and differentiation. Methods of detecting expression or RNA or protein corresponding to kinase genes, anti-apoptotic, cell cycle promoting genes, and genes involved in inhibition of cell cycle progression. Example 4: Plasma Ferritin Concentrations Coreespond to Viral Load and Hypergammaglobulinemia Associated with HIV Infection

Analysis of plasma from HIV-1 infected patients was performed to corroborate the observation that soluble ferritin is present in vivo in the plasma of HTV-infected individuals. These data also show that that the concentration of ferritin is corcelated with the degree of viral replication (i.e., viral load; Fig. 8A) and the increased plasma levels of immunoglobulin (IgA; Fig. 8B). Long-term non- progressors had ferritin levels similar to those of uninfected controls. These individuals also had low levels of IgA. In general, experiments of this type demonstrated a statistically significant correlation between plasma viral RNA load and plasma IgA (pO.Ol), IgG (pO.OOl), and IgM (pO.OOl) in HIV-1 -infected individuals and a highly significant correlation (p<0.001) between plasma viral RNA levels and ferritin concentration. There was also a highly significant correlation between ferritin and plasma IgA (pO.OOOl), IgG (pO.OOOl), and IgM (pθ.0001). These data confirm that ferritin plays a role in B cell dysfunction in HIV-1 infection and that ferritin levels can be used as a prognostic indicator of HIV-infected individuals with an increased likelihood of survival (Savarino et al., 1999, Cell. Biochem. Funct. 17:279-287). In some embodiments, both ferritin and soluble necrosis factor alpha type II receptor (Saves et al., 2001, Clin. Immunol. 99:347-52) are assayed. The correlations between viral load, immunoglobulin synthesis, and ferritin levels can be used in various diagnostic assays.

Example 5: Induction ofB Cell Differentiation and Maturation Elutriated lymphocytes were purified into a population ofB lymphocytes by negative selection with antibody-coupled paramagnetic beads according to the manufacturer's instructions (Dynal Biotech, Oslo, Norway). B cells were also purified by positive selection using antibody to the CD 19 cell surface antigen ofB cells coupled to paramagnetic particles following the manufacturer's instructions (Dynal Biotech, Oslo, Norway). Essentially identical results were obtained with either method of purification. Cell purity was determined to be greater than 96% by flow cytometric staining using fluorochrome-labeled antibodies to CD3 (T cell), CD 19 (B cell), and CD45 (common leukocyte) (BD Pharmingen, San Diego, CA). Cells were cultured at 5 million per ml in RPMI-1640 medium containing 5% fetal bovine serum. Macrophage supematants were prepared as described in Swingler et al., 1999, Nat. Med. 5:997-1003) and were diluted 1 :3 with cell culture medium and added to 2.5 million B cells which where in turn incubated for four days, washed and cultured for an additional seven days in RPMI-1640 medium containing 5% fetal bovine serum. Alternatively, ferritin was added to 2.5 million B cells and the cultures were incubated for four days, washed, and cultured for an additional seven days in RPMI-1640 medium containing 5% fetal bovine serum. Cells were then analyzed by flow cytometry for expression of cell antigens that are expressed during B cell maturation and differentiation. The same protocol was used for the measurement by ELISA of immunoglobulin secretion by purified B cells (see Table 1, Fig. 4, and Fig. 7). slgD is surface immunoglobulin D. As B cells differentiate, immunoglobulin D (IgD) becomes expressed on their surface indicating that the cells are mature. slgD, along with slgM, are the major membrane-bound forms of immunoglobulin expressed by mature B cells and function in the activation ofB cells by antigen. These CD19⁺ antigen naive cells enter the peripheral blood where they can encounter antigen and respond immunologically. If the cells do respond, they proliferate, gain high levels of CD38 surface expression, ultimately lose CD 19 expression, and become plasma cells. Plasma cells are antibody secreting B cells and can be identified by the surface expression of CD 138. The antibody specificity will also undergo affinity maturation during this stage of the immune response. This is a process ofB cell maturation and differentiation. In the primary immune response B cells respond by secreting IgM. Immunoglobulin gene class-switching occurs when subsequent specific accessory signals are provided, i.e., cells rearrange the immunoglobulin heavy-chain C gene segments to express and secrete IgG, IgA, or IgE.

Since ferritin is inducing both B lymphocyte proliferation and secretion of immunoglobulins, ferritin should also be inducing B cell maturation into antibody secreting plasma cells. In experiments designed to test this, B cells were isolated as described supra. Freshly isolated B cells were incubated with or without ferritin (200 ng/ml), cell supernatant (1:3 dilution), or positive control stimulus (pokeweed mitogen was used at 1 :100 dilution as per the manufacturer's instructions; Sigma, St. Louis, MO) cells were then incubated for four days, washed, and cultured for an additional seven days before flow cytometric staining.

In the experiments described herein in which flow cytometry was used to analyze a population ofB cells expressing high levels of CD38 (e.g., as in the experiments whose results are illustrated in Figs. 9A-9C and 1 OA-10C), cells were detected using labeled antibodies as described herein. Maturation was monitored by detection of IgD using antibody specific for human IgD and conjugated to FITC. Some cultures were treated with pokeweed mitogen as a control for B cells expressing high levels of CD38 were first identified by staining with an anti-CD38-cychrome antibody, thus demonstrating one of the steps of maturation. Staining with anti-IgD- FITC also demonstrated maturation by the detection of slgD. Expression of CD138 demonstrated that this population had differentiated to the final plasma cell stage where cells become antibody secreting. In experiments described supra using ELISA analysis, it was shown that soluble immunoglobulin that the plasma cells actively secreted antibody (Table 1 and Figure 7).

The quadrants in Figs. 9A-9C depict flow cytometric analyses and are established by subtracting the background fluorescence from isotype matched fluorescently labeled control antibodies used to stain similarly treated cells. The quadrants delineate cells expressing the marker noted by the x ory axis from non- expressing cells. The four quadrants depicted in Fig. 9A-9C show data as follows: cells detected in the upper left quadrant are positive for expression of the y axis antigen; cells detected in the upper right quadrant are positive for expression of the and the x axis antigens; cells detected in the lower right quadrant are positive for the expression of the x axis antigen, and cells detected in the lower left quadrant are negative for the expression of both and x antigens. Therefore, as illustrated by the data shown in Figs. 9A-9C, ferritin increased the number of mature B cells, i.e., increased numbers of slgD± cells and increased the number of differentiating B cells as shown by the expression of the plasma cell marker CD 138. This confirmation by FACS analysis for ferritin-treated B cells for surface (CD 138) is also confirmed by experiments detecting mtracellular markers (mtracellular IgG present due to active antibody secretion) ofB cells that had matured and differentiated into plasma cells. These data are similar to the experiment described and illustrated in Figure 11 (infra). In the experiments, the data from which are illustrated by Fig. 11 A, the population of CD138⁺ plasma cells was identified directly from the FACS Light Scatter Graph (Side Scatter (SSC) against Forward Scatter (FSC) as shown by the area of 'cells' boxed. This gated region was analyzed for CD 138 expression (SSC against CD138-PE) and positive cells boxed in the R2 gate determined from isotype control antibodies used to stain similarly treated cells. Plasma cells (CD138⁺) that had undergone class switching and were actively secreting IgG were identified by gently permeabilizing the cells after CD138-PE staining and fixation. Intracellular staining was achieved by the addition of fluorescent antibody to IgG in the presence of the permeabilizing agent (CytoFix/CytoPerm™, BD Pharmingen, San Diego, CA). The relative numbers of CD138+/IgG+ plasma cells are shown in Figs. 1 IB and 1 lC.

Consistent with other data described herein, ferritin was demonstrated to drive B lymphocytes down the maturation pathway into antibody secreting plasma cells. In these experiments, B cell maturation is demonstrated by increased numbers of the CD138 antigen-expressing cells. CD38^hl B cells (which are B lymphocytes prepared as described herein and identified by staining with anti-CD38-Cychrome antibody) were analyzed by flow cytometry. These cells showed a greater than seven- fold increase in the number of cells expressing the plasma cell marker CD138 after stimulation with ferritin compared to cells incubated without additions or in the presence of pokeweed mitogen ( 1 : 100 dilution prepared according to the manufacturer's instructions; (Sigma, St. Louis, MO) (Fig. 9C). Ferritin also doubled the number of membrane IgD⁺ cells. Thus ferritin has novel properties that promote B lymphocyte progression in to the maturation and differentiation pathway.

These data demonstrate that ferritin is useful for inducing B cell proliferation and maturation. This property is useful, e.g., for increasing the number ofB cells in a particular clonal line that is useful for production of particular antibodies or for preparing products, e.g., for administration to a subject, of such a cell line. It is also useful for preparing a B cell population that has been expanded and can be selected using methods known in the art for production of specific antibodies. The antibodies can then be prepared for use as a reagent or treatment. Furthermore, the cells prepared in this manner can be introduced into a subject, e.g., a human, in an ex vivo procedure to provide B cells, e.g., B cells that produce a selected antibody. To examine the effect of we/on B cell maturation and differentiation into plasma cells, macrophages were infected with adenovirus expression vectors containing a nef ^'gene (Fig. 10A), or as controls, a GFP gene (Fig. 10B) or were mock infected (Fig. IOC). After culturing the macrophages for 16 hours, supematants were prepared from their culture media and added to B cells which where in turn incubated for four days, washed, and cultured for an additional seven days in the supematants. The B cell cultures were analyzed by flow cytometry after staining with anti-CD38- Cychrome, anti-CD 138-PE, and anti-human IgD-FITC. Supematants from macrophages expressing He/produced the same effect upon B lymphocytes, as did incubation in ferritin (compare Figs. 9A and 10A). These data demonstrate that Nef can be used to induce B cell differentiation and maturation.

Example 6: Immunoglobulin Class Switching induced by Supematants from nef- Producing Cells Experiments were conducted to further investigate the ability of ferritin and supematants from macrophages expressing nef to induce complete differentiation ofB lymphocytes to plasma cells. In these experiments macrophages were infected with adenovirus expression vectors containing a nef ^'gene (Fig. 10A), or as controls, a GFP gene (Fig. 10B), or were mock infected (Fig. IOC). After culturing the macrophages for 16 hours, supematants were prepared from their culture media. The supematants were diluted 1:3 with cell culture medium and added to 2.5 million B cells which were in turn incubated for four days, washed, and cultured for a further seven days in RPMI-1640 medium containing 5% fetal bovine serum. The B cell cultures were analyzed by flow cytometry after staining with anti-CD138-PE. Plasma cells (CD138⁺) that had undergone class switching and were actively secreting IgG were identified by gently permeabilizing the cells after CD138-PE staining and fixation. Intracellular staining was achieved by the addition of anti-IgG-FITC in the presence of the permeabilizing agent (CytoFix/CytoPerm, BD Pharmingen, San Diego, CA). Intracellular expression of IgG on CD 138+ B cell (i.e., plasma cells) demonstrated that these cells had fully differentiated into antibody-secreting B cells and had undergone an immunoglobulin heavy chain C gene segment rearrangement (class switching). Supematants from macrophages expressing «e produced the same effect upon B lymphocytes as did incubation in ferritin.

In a population of the CD138⁺ cells identified after incubation in medium containing ferritin, an increased number of the CD 138+ cells were identified as secreting IgG by intracellular immuno-staining compared to those CD 138+ cells that were in cultures grown without ferritin (Fig. 11C). This is an event that requires immunoglobulin class-switching. The same phenomenon was exhibited by cells incubated in supematants prepared from cells expressing «e/compared to cells grown in supernatant prepared from cells expressing GFP or mock infected. These data demonstrate that ferritin and supematants from macrophages expressing nef are sufficient to induce complete B lymphocyte differentiation to plasma cells. Thus, these data support the finding that ferritin is a hitherto unrecognized B lymphocyte differentiation factor and provide additional evidence that ferritin is useful for induction ofB cell differentiation and maturation. The methods described above can be used assays to identify compounds that decrease ferritin induction ofB cell proliferation. For example, primary B cells isolated as described herein are incubated with ferritin (at range of concentrations from 0 to 1000 ng/ml in 200 ng/ml increments) for four days in the presence or absence of test compounds. Inhibition of cellular proliferation is assessed by incorporation of radioactive thymidine ( H-thymidine) into DNA.

Example 7: Inhibition by Ferritin of the B Cell Antigen-Specific Response

The failure to respond to specific recall antigens is evident in HIV infection. This response relies upon memory cells created when the infected individual had prior antigen challenge. The role of ferritin in this process was investigated. In these experiments, peripheral blood lymphocytes (PBLs) or B lymphocytes were incubated in the presence of ferritin at a range of concentrations from 0 to 800 ng/ml in the presence of the common recall antigen, Tetanus Toxoid (1.3 μ/ml) for four days. Cellular proliferation, a marker for immune recognition and cellular activation, was measured by incorporation of radioactive thymidine (³H-thymidine) into cellular DNA. Inhibition of antigen recognition was determined by comparison with mitogenic stimuli; anti-IgD for B lymphocytes and PHA-P for T lymphocytes. Ferritin-mediated inhibition of the response to the antigen Tetanus Toxoid, which is a thymus independent antigen, did not rely upon T cells for recognition by the immune system. The effect appeared to be ferritin-mediated and so was also analyzed with purified B lymphocytes alone. Comparitors were the B cell mitogen, anti-IgD and an irrelevant protein (bovine serum albumin) in the presence of 0 to 800 ng/ml of ferritin. Cellular proliferation was used as a marker of antigen recognition and measured as described supra. A further positive control for the proliferative capacity of the B lymphocyte preparation was provided by anti-IgD/rCD40L/IL-4.

Ferritin was found to inhibit the antigen-specific response in PBLs and in B lymphocyte cultures. Cellular activation, as assessed by DNA synthesis, was not impaired in PBLs stimulated with the polyclonal T cell mitogen phytohemagglutinin P (PHA-P; Fig. 12C). Neither was DNA synthesis impaired by the mitogenic stimulus provided by the polyclonal B cell activation signal of antibody-mediated cross-linking of membrane IgD (Fig. 12A). However, the specific response to tetanus toxoid was markedly abrogated by ferritin at concentrations that conelate with ferritin levels found in HIV-infected patient plasma (Fig. 12B). The antigen-specific but not mitogenic polyclonal activation was similarly diminished in cultures containing only B lymphocytes (Figs. 13 A and 13B). This function was not previously ascribed to ferritin and further illustrates the harmful consequences of hyperferritinaemia in HIV infection.

These data show that inhibition of ferritin expression or activity can be used to ameliorate effects of HIV infection. Thus, ferritin is a target for identifying compounds that ameliorate such effects, e.g., by inhibiting ferritin expression or activity.

Example 8: Role of Ferritin in B Lymphocyte-Related Cancer

B cell lymphoma is the most prevalent leukemia in HIV infection. Therefore, the effect of exogenous ferritin on the growth of two lymphoma cell lines was examined. Briefly, the Burkitt's lymphoma RAMOS B cell line (ATCC CRL-1596) and the Hodgkin's lymphoma Hs 61 IT B cells (ATCC CRL-7373) were cultured as recommended by the ATCC (www.ATCC.org) in various concentrations of ferritin (0 ng/ml, 250 ng/ml, 500 ng/ml, 1,000 ng/ml, 2,000 ng/ml, or 4,000 ng/ml) for seven days and the increase in cell number was determined as a percentage of the number of cells in control cultures that were not incubated with ferritin.

An increase in cell number of 30 to 35% was measured in both cell lines after seven days in culture (Tables 6 and 7). The RAMOS B cell line was also found to secrete IgG in a linear response to ferritin concentration (Fig. 14). The immunoglobulin content of cell-free cell culture supematants was determined by ELISA. IgG was found to be secreted in a dose-dependent response manner to exogenously added ferritin. Five million RAMOS cells were incubated in with 0- 2000 ng/ml ferritin for four days, washed and incubated for an additional five days before immunoglobulin release was measured.

Table 6.

Burkitt's Lymphoma RAMOS B Cells

Table 7.

Hodgkin's Lymphoma Hs 61 IT B cells

These findings demonstrate that ferritin can also play a role in certain types of cancer in addition to the pathogenesis of HIN infection and B cell immunology. Thus, treatments that inhibit the expression or activity of ferritin are useful for treating cancers in which ferritin induces undesirable cell proliferation. Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of increasing at least one ofB lymphocyte proliferation, differentiation, or maturation, the method comprising administering ferritin or a compound that increases ferritin expression or activity to a B lymphocyte or a B lymphocyte precursor cell in an amount sufficient to increase B lymphocyte proliferation, differentiation, or maturation.

2. The method of claim 1, wherein the compound is Nef or an active fragment thereof.

3. The method of claim 1, wherein the compound is administered to a cell in vitro.

A. The method of claim 1, wherein the compound is administered to a plurality of cells.

5. The method of claim 1, wherein ferritin is administered.

6. The method of claim 5, wherein the ferritin comprises the L subunit of ferritin.

7. The method of claim 5, wherein the ferritin is a recombinant L subunit of ferritin.

8. The method of claim 1, wherein the production of at least one of IgG, IgA, or IgM is increased.

9. The method of claim 1, wherein the compound is administered to a subject.

10. The method of claim 9, wherein the subject is a human.

11. The method of claim 1, wherein the cell is from a subject that has Burkitt's lymphoma or Hodgkin's lymphoma.

12. The method of claim 1, wherein the compound is administered to a cell in vitro.

13. The method of claim 1, wherein the compound is administered to a cell that has been genetically engineered.

14. The method of claim 1, wherein the compound is administered to a cell that has been genetically engineered to express an antibody.

15. A method for identifying a compound that inhibits the activity of ferritin on a lymphocyte, the method comprising,

(a) obtaining a cell sample containing a lymphocyte;

(b) incubating the cell sample in the presence of ferritin and a test compound; and

(c) determining whether the test compound modulates a ferritin-associated effect on the lymphocyte in the presence of ferritin compared to a cell sample incubated in the absence of the test compound and in the presence of ferritin.

16. The method of claim 15, wherein the compound decreases the ferritin-associated effect on the lymphocyte.

17. The method of claim 15, wherein the compound increases the ferritin-associated effect on the lymphocyte.

18. The method of claim 15, wherein the compound decreases the ferritin-associated effect on the lymphocyte and decreases the expression or activity of NF-κB, BCL-

2, Casper/c-FLTP, or cyclin Dl.

19. The method of claim 15, wherein the compound increases the ferritin-associated effect on the lymphocyte and increases the expression or activity of NF-κB, BCL- 2, Casper/c-FLTP, or cyclin Dl .

20. The method of claim 15, wherein the compound increases the ferritin-associated effect on the lymphocyte and decreases expression or activity of a cell cycle progression protein.

21. The method of claim 20, wherein the cell cycle progression protein is p27^KJP1 or p53.

22. The method of claim 20, wherein the compound does not affect MEK1 expression or activity.

23. The method of claim 15, wherein the ferritin-associated effect on the lymphocyte is at least one of the following: cellular proliferation, differentiation into a plasma cell, increased secretion of non-specific immunoglobulin, increased secretion of IgG increased secretion of IgM, secretion of IgA, increased secretion of non- specific immunoglobulin, increased expression of IgG increased expression of

IgM, and increased secretion of IgA.

24. The method of claim 15, wherein the lymphocyte is a peripheral blood monocyte or a B lymphocyte.

25. A method of decreasing B lymphocyte proliferation, differentiation, or maturation, the method comprising contacting a B lymphocyte or precursor of a B lymphocyte with compound that inhibits ferritin expression or activity in an amount sufficient to decrease B lymphocyte proliferation, differentiation, or maturation.

26. The method of claim 25, wherein the compound inhibits expression or activity of Nef.

27. The method of claim 25, wherein the compound inhibits NF-κB expression or activity.

28. The method of claim 25, wherein the compound does not affect MEK1 expression or activity.

29. The method of claim 25, wherein the compound is administered to a cultured cell.

30. The method of claim 25, wherein the compound is administered to a B lymphocyte or precursor of a B lymphocyte in a subject.

31. The method of claim 30, wherein the subject is a human.

32. The method of claim 25, wherein the cell is from a subject who has Burkitt's lymphoma or Hodgkin's lymphoma.

33.. A method of inhibiting immunoglobulin expression in a B cell, the method comprising

(a) providing a B cell; and

(b) contacting the cell with an inhibitor of ferritin expression or activity.

34. The method of claim 33, wherein the B cell is in a mammal.

35. The method of claim 33, wherein the cell is in vitro.